The constitutive androstane receptor (CAR, NR1I3) is a member of the nuclear receptor superfamily that is expressed primarily in the liver. CAR plays a key role in regulating all three phases of biotransformation, pathways that determine the fates of metabolism and toxicity associated with exposures to drugs, other xenobiotics, and endogenous substances such as bile acids, thyroid hormone, heme, and steroids. Due to the importance of this receptor system as a determinant of xenobiotic disposition in man, together with CAR's increasingly recognized role as a physiological regulator, it is imperative to fully characterize the biological processes directed by the human CAR proteome. Progress in the previous research program included the discovery of novel forms of CAR, CAR2 and CAR3; receptors generated through the use of alternative splicing mechanisms that result in the insertion of 4- and 5- amino acids, respectively, within the receptors' ligand binding domain. These receptors are expressed simultaneously with CAR in human livers, but possess highly unique functional attributes. Unlike CAR itself, the variant CARs are ligand-activated receptors, exhibit selective chemical activation profiles, and appear to differentially modulate target gene expression. We hypothesize that alternative splicing enhances the functional diversity of CAR molecules, resulting in overlapping, yet discriminating roles as xenobiotic sensors, driving the interplay of hepatic gene expression networks that in turn, provide critical integration signals directing human physiological responses to the chemical milieu. To test our hypotheses, a progression of three specific aims will be deployed that include: 1) the structural analysis of crystallized CAR proteins; 2) use of primary human hepatocyte culture models to enable receptor-driven transcriptional profiling, target gene interactions, and assessment of the dynamics of receptor intracellular localization; and, 3) humanized transgenic mouse investigations to analyze the transcriptional controls modulated by the receptors in vivo, and the phenotypic impact of receptor expression within a series of serum markers. We predict that the respective modulation of genomic programs and the resulting signaling circuitry controlled by the variant CARs have critical functional implications for human health, determining outcomes such as drug-drug interactions, drug and bile acid induced hepatotoxicity, carcinogen and steroid metabolism, and the regulation of lipid and energy homeostasis. The results of the proposed research will contribute important new advances and insights regarding the role of the CAR nuclear receptors as direct modulators of the dynamic gene regulatory networks that determine toxicological and physiological responses to both xenobiotic and endogenous substances.